Process of producing gluconic acid and gluconates

ABSTRACT

ALDONIC ACIDS AND ALDONATES, MORE PATRICULARLY, GLUCONIC ACID AND GLUCONATES, ARE PREPARED BY A FERMENTATION PROCESS IN WHICH A PORTION OF THE ALDOSE IS FERMENTED. THEREAFTER MORE ALDOSE IS ADDED AND FERMENTED UNTIL THE FERMENTATION MEDIUM CONTAINS A HIGH CONCENTRATION OF ALDONIC ACID AND/OR ALDONATE.

United States Patent O1 lice 3,576,7 l8 Patented Apr. 27, 1971 Int. Cl.C12d 1/06 US. Cl. 195-36 13 Claims ABSTRACT OF THE DISCLOSURE Aldonicacids and aldonates, more particularly, gluconic acid and gluconates,are prepared by a fermentation process in which a portion of the aldoseis fermented. Thereafter more aldose is added and fermented until thefermentation medium contains a high concentration of aldonic acid and/oraldonate.

This application is a division of application Ser. No. 432,378 filedFeb. 12, 1965, now US. Pat. No. 3,454,501.

This invention relates to aldonic acids and aldonates and moreparticularly to new gluconic acid and gluconate compositions whichexhibit highly desirable properties, and processes for the production ofthese compositions.

Gluconic acid is usually made commerciall from glucose either bychemical oxidation or by fermentation processes. The free acid isusually sold as a 50% by weight solution in water. The salts such assodium gluconate or ammonium gluconate are sold as solids but aredissolved in water before use in processing operations. The presentinvention is concerned with a fermentation process for making gluconicacid and gluconates, and especially with a process for preparing new anduseful liquid mixtures of gluconic acid and gluconates.

The value of gluconic acid and gluconates in commerce is well known, asare the various gluconic acid and gluconate products adapted for theseuse and processes for obtaining them. However, the available productsand processes suffer from disadvantages which are desirably overcome.One important problem concerns the economical fermentation production ofhigh concentrations of gluconic acid and gluconates. Another problem isthe difiiculty of processing concentrated solutions of gluconic acidsalts. Still another problem is the preparation of stable concentratedgluconate liquid compositions. A further problem is the economicalconversion of gluconates to gluconic acid.

It is therefore an object of this invention to provide an economicalprocess for the submerged culture fermentation production of highconcentrations of gluconic acid and gluconates.

Another object is to provide a simple and reliable procedure forprocessing concentrated solutions of gluconates.

An additional object is to provide for the production of stableconcentrated gluconic acid and gluconate liquid compositions.

A further object is to provide an economical process for the conversionof gluconates to gluconic acid.

Other objects include the simple, reliable and economical production ofgluconic acid and gluconates of a high degree of purity. These and otherobjects and advantages of the invention will be apparent upon referenceto the following description.

In accordance with one embodiment of the invention, new and usefulmethods have been discovered for conducting the gluconic acid submergedculture fermentation in concentrated medium solutions, such mediumsolutions containing glucose, or its equivalent as hydrolyzed starches,dextrins, syrups and the like, in concentrations far in excess of thosepreviously known in the art. For example, the prior art refers to mediumsolutions containing up to 40% glucose, with the preferred range beingconsiderably less than this concentration. In this invention, it isroutine to ferment and convert fermentation medium solutions to whichhave been added in excess of 40% glucose with the preferred additionbeing the equivalent of approximately 50-75% glucose, and to obtain anend product, as for example sodium gluconate or its equivalent, indesirable concentrations in excess of 60% in the final medium solution.Although high glucose concentrations, particularly in the 5075% range,are not readily fermented, it has been found in the practice of thisinvention that increased concentrations of glucose can be readilyutilized and converted to glusenic acid if the fermentation medium isprepared with a portion of the glucose and the remainder of the glucoseadded later during the fermentation process. In other words, the glucoseis added in increments or aliquots. For example, it has been found thatcomplete fermentation and conversion to gluconic acid can be obtained byinitially fermenting a nutrient solution containing approximately 28%glucose and subsequently adding and fermenting approximately 30%additional glucose. Likewise, glucose combinations of approximately 27%,30%, 8% and 9%; 34% and 26%; 42% and 21%; 41%, 15% and 10%; and the likecan be readily employed. Alternatively, the glucose can beintermittently added in smaller aliquots. One additional advantage ofthis process is that it is only necessary to sterilize the initialfermentation glucose medium as the subsequent portions of glucose can beadded without sterilization, resulting in an operating economy. Further,the advantageous use of non-sterilized glucose results in a desirablylighter colored fermentation medium and, subsequently, in a desirablylighter colored end product.

During at least the initial course of the fermentation, a base, forexample sodium hydroxide, potassium hydroxide, ammonium hydroxide, orthe like, is added to the fermentation medium to neutralize the gluconicacid being produced. Although the prior art emphasizes the importance ofcompletely neutralizing all of the gluconic acid as it is formed, it hasbeen found that this is not a requirement of the present process asherein described and that it is usually desirable not to completelyneutralize all the gluconic acid formed. While the acid neutralizationcan be continued until the fermentation is completed and all of theglucose has been converted to gluconic acid, it is one of the-featuresof this invention that the neutralization of the gluconic acid becontinued only until the organism has produced substantially optimumamounts of cell growth and glucose oxidase, which enzyme is involved inthe oxidation of glucose to gluconic acid. At approximately this point,the neutralization of the gluconic acid being produced is stopped, andthe fermentation continued without neutralization until substantiallyall of the glucose added is converted to gluconic acid.

Alternatively, the neutralization of the gluconic acid being producedcan be stopped at an earlier period or at a later period. For example,in one embodiment of the invention, the neutralization of the gluconicacid is stopped after approximately 28% glucose has been converted toacid, and the fermentation is then continued as an additional 30%glucose is added and converted to acid without any furtherneutralization. As another alternative, the neutralization of thegluconic acid can be stopped at another point, as for example afterapproximately 42%' glucose has been converted to acid, and thefermentation then continued as an additional 21% glucose is added andconverted to acid without any further neutralization. Thus, thepercentage of gluconic acid and neutralized gluconates produced in thefermentation can be readlly varied depending on the requirements of theprocess as later described.

This process advantageously results in compositions of mixtures ofsubstantial amounts of gluconic acid and neutralized gluconate salt,such processes and compositions having many desirable features. Forexample, the neutralization of only a portion of the gluconic acidproduced during the fermentation results in a considerable economy ofbase used, when the resultant composition is used as such. In addition,the reduced amount of base used desirably results in decreased totalsolution solids, a feature technically important in achieving optimumagr tation-aeration conditions during the fermentation. Further, thereduced amount of base used desirably results in decreased solutionviscosities, a feature technically important during the recoveryprocessing operation.

When the fermentation is carried out for the production of completelyneutralized sodium gluconate, the salt is present in the final wholeculture medium in excess of its solubility level. Thus, it is necessaryto heat the whole culture to redissolve the sodium gluconate prior tothe filtration operation and to keep the mixture warm during thisoperation. The solution solubility of sodium gluconate becomes even morecritical during the subsequent handling and concentration procedureprior to the crystallization and drying operations, when there isincreased danger of the concentrated supersaturated sodium gluconatesolution crystallizing out and solidifying in the transfer lines andprocessing equipment. While this inherent danger can usually be avoidedby keeping the concentrated supersaturated sodium gluconate solution hotand by processing the solution as rapidly as possible, these conditionsare not always completely possible or economical. In addition, it isdesirable not to heat the sodium gluconate solution unnecessarily asheating increases the color of the solution and subsequently the colorof the end product. Further, residual sodium gluconate crystalline seedin a transfer line may cause a concentrated supersaturated solutionsubsequently passing through the line to prematurely crystallize out andsolidify. It is evident that these and other similar technical problemsare desirably overcome.

It is an important feature of this invention that these and similartechniml problems are readily and simply corrected. For example, inaccordance with one embodiment of the invention desirable compositionsof mixtures of gluconic acid and neutralized gluconates can be produced,as for example neutralized sodium gluconate, in the final medium in theratio of approximately 0.5-3 parts gluconic acid to 1 part neutralizedsodium gluconate. These compositions and similar other compositions areunique in that they are readily soluble over a wide range ofconcentrations, concentrations in excess of the levels at whichneutralized sodium gluconate solutions would quickly crystallize andsolidify. Thus, in accordance with this invention, it is possible toconcentrate the composition to the desired high levels without anydanger of crystallization or solidification. Further, the solubleconcentrated solution can be safely and conveniently transferred to thedryer feed tank where it is completely neutralized with sodium hydroxideor similar base and the product recovered by drying as known to the art.Alternatively, the final processed fermentation medium filtrate can betransferred directly to the dryer feed tank without any furtherconcentration, where it is completely neutralized and recovered bydrying. In this manner, it is possible in accordance with this inventionto obtain a sodium gluconate dry product of 98% purity with a veryadvantageous operating economy. If desired, the soluble concentratedsolution can be easily and routinely transferred to the crystallizertank 'where it is completely neutralized with sodium hydroxide orsimilar base, the crystallization allowed to proceed and the sodiumgluconate crystals recovered and dried as known to the art. In thismanner, it is possible in accordance with the invention to obtain asodium gluconate dry product of purity. These process improvements thusadvantageously result in a very desirable versatility and operatingeconomy.

Except for gluconic acid, the only commercially available liquidcomposition, the salts of gluconic acid are produced on large scale onlyas dry solid products. One reason for this is the relative poorsolubility of the gluconates. For many technical purposes, however, itwould be very desirable to have other liquid products available as suchpreparations readily lend themselves to large scale operation.Unfortunately, it is difiicult to prepare and maintain such liquidconcentrate products from gluconates. It is therefore one of the objectsof this invention to provide for the preparation of stable gluconic acidand gluconate liquid concentrate compositions which can readily be usedin place of the unstable concentrated gluconate solutions. It is afurther object of this invention to provide compositions havingsolubility properties and other advantages which are useful anddesirable, especially when compared to the commercially available liquidgluconlc acid product, an important use of which is for the preparationof alkaline formulations. For example, the gluconic acid and gluconateliquid compositions require less base than the gluconic acid product forthe preparation of alkaline formulations. In addition, one of thedisadvantages of the liquid gluconic acid product is that it is producedonly as a 50% by weight composition which should be stored attemperatures above 7 C. (45 F.) to prevent crystallization. Further,when the liquid gluconic acid product is prepared at desired higherconcentrations, the danger of crystallization is markedly increased.

These are disadvantages that are desirably overcome and it has beendiscovered that this can be easily and economically accomplished usingcompositions of gluconic acid and gluconates or their equivalent. Forexample, it has been found that liquid compositions such as gluconicacid plus sodium gluconate, gluconic acid plus potassium gluconate,gluconic acid plus ammonium gluconate, gluconic acid plus sodiumgluconate plus ammonium gluconate, gluconic acid plus sodium gluconateplus potassium gluconate plus ammonium gluconate, and the like can beprepared in high concentration, as for example as high as approximately5090% by weight or its equivalent, with the desired stability andsolubility properties. In addition to producing these compositions bydirect formulation, it has been found that, as previously de scribed,one can very advantageously produce the com positions directly in thefermentation, or, alternatively, produce one form of the compositiondirectly in the fermentation and subsequently modify it by directformulation.

One additional advantage of this invention is in regard to theeconomical production of gluconic acid. In the prior art processes forthe production of this product, the procedures include theneutralization of the reaction mixture or fermentation medium withcalcium base or the like, for the important technical function ofprecipitating the acid as its insoluble salt. Such a procedure, however,requires the subsequent relatively expensive chemical conversion of therecovered insoluble salt to free gluconic acid by acidification withsulfuric acid. Further, the necessary use of large amounts of calciumbase to completely neutralize the gluconic acid makes it very diflicultto recover the acid by any other procedure. It has been discovered thatan embodiment of this invention, whereby the fermentation is carried outusing a minimum amount of base, for example sodium hydroxide, topartially neutralize the gluconic acid, lends itself to the relativelyinexpensive recovery of gluconic acid from the final fermentationmixture. For example, the sodium cation present in a typical finalfermentation mixture containing approximately 2 parts gluconic acid and1 part sodium gluconate can be conveniently removed by ion exchangeresin treatment, dialysis, electrodialysis, etc. This method for theproduction of gluconic acid has the further advantage in that it can becarried out directly using the final processed fermentation filtrate,eliminating the need for expensive precipitation and conversionprocedures. In this manner, it is possible in accordance with thisinvention to obtain a gluconic acid product of 98-100% purity with avery advantageous operating economy.

The following examples are illustrative of the methods and compositionsaccording to the invention. It is to be understood that the invention isnot limited to the examples nor to the particular materials,proportions, conditions and procedures set forth therein. Allpercentages here are on a weight:volume basis unless otherwiseindicated. For example, 40% means 40 grams per 100 milliliters of totalsolution.

EXAMPLE 1 (a) A nutrient medium 'was prepared from the followingmaterials:

Corn meal 10 Dextrose monohydrate 20 Corn steep liquor 50 Water to 1000ml.

After adjusting the mixture to pH 7.0-7.2 with sodium hydroxide, 5 gramsof calcium carbonate were added. A slant culture of the gluconicacid-producing organism Aspergillus niger, N.R.R.L.3 was used toinoculate a number of 250 ml. Erlenmeyer flasks, each containing amixture of 5 grams of wheat bran and 5 ml. of the above medium, themixture previously sterilized for 30- 45 minutes at 121 C. These flaskswere incubated at 28 C. for 5-15 days until good growth and sporulationwere obtained.

solution and inoculated with one flask of the inoculum prepared in (a)above slurried in 2 liters of sterile water. The organism was thencultivated at 33 C. under submerged conditions of aeration for a periodof approximately 15-22 hours.

Grams 6 (c) A nutrient medium was prepared from the following materials:

1 Commercial dextrose monohydrate was used. For clarity,

values are recalculated as pure dextrose and the amounts used areindicated in the individual examples. After adjusting the pH toapproximately 4.5 with sulfuric acid, the mixture was sterilized withsteam for 30 minutes at 121 C. and cooled to .33 C. The medium was thenadjusted to pH 6.4-6.8 with sterile sodium hydroxide solution andinoculated with 10 gallons (1 0%) of the inoculum prepared as describedin (b) above. The organism was then cultivated at 33 C. under submergedconditions of aeration and agitation for the indicated period of hours,as shown in the individual examples.

(d) The data shown in the following Table I are for a series of runscarried out with varying glucose concentrations and the fermentationmedium pH controlled in the approximate range of 6.2-6.6 by theautomatic addition of sterile 50% by weight sodium hydroxide solution.As indicated by the data, the glucose in the 28% and 42% runs wasessentially completely utilized with excellent conversion to gluconicacid. Increasing the percent glucose in the medium, especially to 63%,resulted in slower utilization rates.

TABLE I Run Initial percent glucose 27. 7 42.0 63. 1 pH control Yes YesYes Fermentation time, hours 23 48. 6 l 71 Percent residual glucose 0.230.07 36. 9 Average glucose utilization, percent /hr 1. 22 0. 0. 4 FlnalpH 7. 0 7. 0 6. 4 Percent gluconate 2 27. 4 40. 2 19. 4 Percentconversion from glucose 94. 3 94. 3 28. 2

1 Not complete. 2 Assayed by polarimeter.

(e) To illustrate the importance of initial pH control in thisfermentation, an identical run (41.5% glucose) was carried out exceptthat the medium pH was not controlled and sodium hydroxide was notadded. As indicated by the data in the following Table II, only about25% of the glucose was utilized and only a portion of this was convertedto gluconic acid, despite the fact that the mycelium solids valueindicated that better than normal fungal growth had been obtained.

1 Titration value calculated as gluconic acid.

EXAMPLE 2 The procedures of Example 1 were repeated using a mediumcontaining 27.4% glucose and the pH maintained at 6.4 by the automaticaddition of 50% by weight sodium hydroxide. After the completion of thefermentation (cycle No. 1), additional glucose (non-sterile) 'was addedto the medium so that the concentration was 30.1% and the fermentationcontinued with the pH maintained as before. After the completion of thefermentation (cycle No. 2), additional glucose (non-sterile) Was againadded to the medium so that the glucose concentration was 7.8% and thefermentation continued as before. After the completion of thefermentation (cycle No. 3) additional glucose (non-sterile) was againadded to the medium so that the glucose concentration was 8.7% and thefermentation continued as before. The data for these fermentation cyclesare shown in the following Table III. As indicated by these data,essentially complete utilization and excellent conversion of the glucoseto sodium gluconate was achieved, with the final sodium gluconateconcentration being 68.2%. Considerable crystallization occurred in thefermentation medium at these high sodium gluconate levels and by the endof fermentation cycle No. 4, the precipitated sodium gluconate occupiedapproximately 40% of the final medium volume on settling. It wasnecessary to heat the final medium to approximately 70 80 C. Toredissolve the precipitated gluconate, and to hold the medium at thistemperature range during the filtration operation. A clear filtrate wasobtained in this manner; however, the sodium gluconate quicklyreprecipitated as soon as the filtrate temperature dropped appreciably.

TABLE III Fermentation cycles Initial percent glucose 27. 4 30. 1 7. 88. 7 pH control Yes Yes Yes Yes Fermentation time, hours- 26 20 13 38Percent residual glucose 0. 0 05 0.0 0.65 Final pH 6. 4 6.4 6.4 7.0Percent sodium gluconate 26. 4 52. 2 58. 0 6. 82 Percent conversion fromglucose 93. 2 95. 4 98. 6 98. 0

l Assayed by polarimeter.

EXAMPLE 3 The procedures of Example 2 were repeated using a mediumcontaining 40.9% glucose and the medium pH maintained at 6.6 with 50% byweight sodium hydroxide. After the completion of the fermentation (cycleNo. 1), additional glucose (non-sterile) was added to the medium so thatthe concentration of glucose was 15.2% and the fermentation continuedwithout the addition of any base. After the completion of thefermentation (cycle No. 2), additional glucose (non-sterile) was againadded to the medium so that the concentration of glucose was 9.9% andthe fermentation continued without the addition of any base. The datafor these fermentation cycles are shown in the following Table IV. Asindicated by these data, essentially complete utilization and excellentconversion of the glucose to gluconic acid and sodium gluconate wasachieved, with the final yield, calculated as sodium gluconateequivalent, being 62.1%. The final fermentation medium contained thegluconic acid and sodium gluconate completely in solution, and wasprocessed by filtration at room temperature to give a clear lightcolored filtrate which remained clear on standing.

TAB LE IV Fermentation cycles Initial percent glucose 40. 9 15. 2 9. 9pH control Yes No No Fermentation time, hours 45 12 15 Percent residualglucose--- 0. 14 0.47 0. 00 Final pH 6. 6 4. 0 3. 8 Percent sodiumgluconate equivalent 39. 1 53. 2 62. 1 Percent gluconic acid 2 0. 0 16.2 25. 8 Ratio gluconic aeid:sodium gluconate 0. 46 0.77 Percentconversion from glucose 96. 1 99.0 100 1 Neutralized sample assayed bypolarimeter. a Titration value calculated as gluconic acid.

8 EXAMPLE 4 (a) The fermentation cycle procedures of Example 3 wererepeated for a series of 2-cycle runs whereby the, initial fermentationcycle was maintained at pH 6.4 bythe addition of sodium hydroxide andthe subsequent fermentation cycle was carried out without the additionof any base. The initial fermentation medium in each run containedapproximately 42% glucose; however, the

addition of sodium hydroxide in the individual runs was discontinued atdifferent times so as to vary the amount of gluconic acid produced inthe fermentations. As shown by the data in the following Tables V, VI,VII, and VIII, the sodium hydroxide addition was stopped afterapproximately 42%, 37%, 33%, and 26% glucose had been utilized in therespective runs. In each case, the same amount of glucose (non-sterile)was then added to each medium and the fermentations continued withoutthe addition of any base. As indicated by the data, es-

sentially complete utilization and excellent conversion of the glucoseto gluconic acid and sodium gluconate was achieved, with the yields,calculated as sodium gluconate equivalent, being 60.8%, 61.1%, 62.4%,and 60.2%,-

TAB LE V Fermentap tion cycles Initial percent glucose 42.0 21. 3 pHcontrol Yes No Fermentation time, hour 48. 5 23 Percent residual glucose0. 07 0. 0 Final pH 6. 4 3. 7 Percent sodium glucon 40. 2 60. 8 Percentgluconic acid 2 0. 0 23. 9 Ratio gluconic acid: sodium gluconate- 0.70Percent conversion from glucose 94. 4

1 Neutralized sample assayed by polarimeter. 2 Titration valuecalculated as gluconic acid.

TABLE VI Fermentation cycles Initial percent glucose 42. 0-5. 1 25. 7 pHcontrol Yes No Fermentation time, hours. 46. 5 26 Percent residualglucose-.. 5. 1 0.0 Final pH 6. 4 3. 5 Percent sodium gluconateequivalent 35. 1 61. 1 Percent gluconic acid 2 0. 0 28. 5 Ratio gluconicacidzsodium gluconate 0. 96 Percent conversion from glucose 95. 5 97. 8

l Neutralized sample assayed by polarimeter. Z Titration valuecalculated as gluconic acid.

TABLE VII Fermentation cycles Initial percent glucose 42. 0-9. 0 28. 5pH control Yes No Fermentation time, hour 42. 5 33. 5 Percent residualglucose 9. 0 0. 02 in 6. 4 3. 55 Percent sodium glucon 30. 0 62. 4Percent gluconic acid 2 0. 0 32. 9 Ratio gluconic aeidzsodiumgluconate 1. 28 Percent conversion from glucose 92. 2 97. O

1 Neutralized sample assayed by polarimeter. Z Titration valuecalculated as gluconic acid.

1 Neutralized sample assayed by polarlmeter. Z Titration valuecalculated as gluconic acid.

(b) The fermentation cycle procedures of Example 3 were repeated for aseries of 2-cycle runs with varying glucose concentrations whereby theinitial fermentations were maintained at pH 6.4 by the addition ofsodium hydroxide and the subsequent fermentations were carried outwithout the addition of any base. As shown by the data in the followingTables IX and X, essentially complete utilization and excellentconversion of the glucose to gluconic acid and sodium gluconate wasachieved, with the yields, calculated as sodium gluconate equivalent,being 60.3% and 59.5%. The gluconic acidzsodium gluconate ratios were1.68 and 1.21, respectively. The final fermentation medium in each casecontained the gluconic acid and sodium gluconate completely in solutionand was processed by filtration at room temperature to give a clearlight colored filtrate which remained clear on standing.

TABLE IX Fermentation cycles Initial percent glucose 27. 7 80. pHcontrol Yes No Fermentation time, hours 23 45 Percent residualglucose.-.. 0. 0 Final pH 3. 4 Percent sodium gluconate equivalent 1 27.4 60. 3 Percent gluconic acid 0.0 35. 4 Ratio gluconic acidzsodlumgluconate. 1. 68 Percent conversion from glucose 94. 0 99.0

1 Neutralized sample assayed by polarimeter. 2 Titration valuecalculated as gluconic acid.

TABLE X Fermentation cycles Initial percent glucose 34. 3 26. 0 pHcontrol Yes No Fermentation time, hours 35 23 Percent residualglucose 1. 7 0. 0 Final pH 6.4 3. 45 Percent sodium gluconate equivalent31. 9 69. Percent gluconic acid 0.0 30. 7 Ratio gluconic acidzsodiumgluconate 1. 21 Percent conversion from glucose 94.5 98. 5

1 Neutralized sample assayed by polarimeter. 2 Titration valuecalculated as gluconic acid.

EXAMPLE 5 The final fermentation medium usually contains small amountsof sulfate, phosphate, oxalate, etc. anions which are desirably removed,and this can be conveniently accomplished by treating the medium orsolution with small amounts of calcium carbonate, calcium hydroxide, orthe like to precipitate the anions as insoluble salts.

(a) The final solution from a fermentation carried out according to thegeneral procedure of Example 4 and containing 40.0% gluconic acid and62.8% sodium gluconate equivalent (gluconic acidzsodium gluconate ratio=2.l7) tested positive for sulfate, phosphate, oxalate, etc. It wasslurried with calcium carbonate (1%) and diatomaceous earth (2%) andfiltered on a wet diatomaceous earth pre-coated filter. The clearfiltrate, diluted by water from the pre-coat filter, contained 36.3%gluconic acid and 60.4% sodium gluconate equivalent (gluconicacid:sodium gluconate ratio=1.81) and tested negative for sulfate,phosphate, oxalate, etc.

(b) Final solution from a fermentation carried out according to thegeneral procedure of Example 4 and containing 35.0% gluconic acid and58.8% sodium gluconate equivalent (gluconic acidzsodium gluconateratio=1.76) tested positive for sulfate, phosphate, oxalate, etc. It wasslurried with calcium hydroxide (0.74%) and diatomaceous earth 1%) andfiltered of a wet diatomaceous earth pre-coated filter. The clearfiltrate, diluted by wash and the water from the pre-coat filter,contained 25.7% gluconic acid and 48.5% sodium gluconate equivalent(gluconic acidzsodium gluconate ratio=l.29) and tested negative forsulfate, phosphate, oxalate, etc.

EXAMPLE 6 (a) Clear filtrate from a fermentation carried out andprocessed according to Examples 4 and 5 containing 34.4% gluconic acidand 59.1% sodium gluconate equivalent (gluconic acidzsodium gluconateratio=l.65) was neutralized to pH 7.5 with 50% by weight aqueous sodiumhydroxide and the resultant mixture dried on an atmospheric double drumrotary drier (60 lbs. per sq. inch steam pressure). An off-white sodiumgluconate solid of 98.4% purity was obtained.

(b) Clear filtrate from a fermentation carried out and processedaccording to Examples 4 and 5 containing 26.1% gluconic acid and 58.0%sodium gluconate equivalent (gluconic acidzsodium gluconate ratio=0.90)was neutralized to pH 7.5 with 50% by weight aqueous sodium hydroxideand the resultant mixture dried on an atmospheric double drum rotarydrier (60 lbs. per sq. inch steam pressure). An off-white sodiumgluconate solid of 98.4% purity was obtained.

(0) Clear filtrate from a fermentation carried out and processedaccording to Examples 4 and 5 and containing 36.3% gluconic acid and60.4% sodium gluconate equivalent (gluconic acidzsodium gluconateratio=1.81) was concentrated in vacuo to 91.6% sodium gluconateequivalent. The concentrate at this point was sparkling clear andremained clear at room temperature. The clear concentrate was thenneutralized to pH 7.4 with 50% by weight sodium hydroxide and theresultant sodium gluconate crystalline slurry dried on an atmosphericdouble drum rotary drier (60 lbs. per sq. inch steam pressure). Anoff-white sodium gluconate solid of 98.3% purity was obtained.

(d) Clear filtrate from a fermentation carried out and processedaccording to Example 4 and containing 23.9% gluconic acid and 60.7%sodium gluconate equivalent (gluconic acid; sodium gluconate ratio=0.70)was concentrated in vacuo to 89% sodium gluconate equivalent. Theconcentrate at this point was sparkling clear and remained clear at roomtemperature. The clear concentrate was then neutralized to pH 7.2-7.3with 50% by weight sodium hydroxide and the resultant sodium gluconatecrystalline slurry dried on an atmospheric double drum rotary drier(60-65 lbs. per sq. inch steam pressure). An off-white sodium gluconatesolid of 97.0% purity was obtained.

EXAMPLE 7 Liquid compositions of gluconic acid and gluconic acid plusgluconates were prepared by formulation and from fermentation processedfiltrates according to the schedule shown in the following Table XI.These compositions were concentrated in vacuo.to 90% by weight solidsand diluted to the indicated concentration levels with water. Thediluted solutions were then stored at 5-7 C. (4l45 F.) and examinedperiodically for evidence of crystallization. The data obtained areshown in the following Tables XII to XXV. As indicated by these data,the gluconic acid plus gluconate liquid com- TABLE XIV.COMPOSITIONObservations after indicated days at 7 C.

5 Concentration, percent:

1 1 positions, with the exception of the gluconic acid plus sodiumgluconate 0.67 ratio composition, were essentially unaffected under thestorage conditions. In contrast, the gluconic acid liquid compositionwas not soluble over a wide concentration range. Further, the gluconicacid plus mixed gluconate compositions, as for example, gluconic acidplus sodium gluconate plus ammonium glu- LLLLLLLLL LLLLLLLLL LLLLLLLLLLLLLLLLLL LLLLLLLLL LLLLLLLLL conate and gluconic acid plus sodiumgluconate plus p0- tassium gluconate plus ammonium gluconate, at theequivalent 0.67 ratio composition, were completely soluble at theconcentration levels at which the gluconic acid plus sodium gluconate0.67 ratio composition was not soluble.

salt Refer to 1. 33 Table XVI.

1 Table XVII. 0. 67 Table XVIII. 1. 64 Table XIX.

0.91 Table XX.

2 Table XXI. 2 Table XXII. 2 Table XXIII. 0. 67 Table XXIV.

0. 67 Table XXV.

Observations after indicated days at 57 C.

Ratio gluconic acid,

gluconate TABLE XV.COMPOSITION D Concentration, percent:

do do do do. Filtrate from a fermentation carried out and processedaccording to Examples 4 and 5. Gluconic acid, sodium hydroxide-.-Gluconic acid, potassium g Gluconic acid, ammonium M Gluconic acid,sodium gluconate, am-

tassium hydroxide, ammonium hydroxide.

TABLE XI Prepared from- Glugonic acid, sodium gluconate- O out andprocessed according to Examples 4 and 5. .do. Filtrate from afermentation carried luconato Gluconic acid, potassium hydroxidegluconate. Gluconic acid, ammonium hydroxide- Gluconic acid, sodiumgluconate, am-

monium hydroxide. Giuconic acid sodium gluconate, po-

Composition Gluconic acid-. Giuconic acid, sod

do. do. do

J- .do

monium gluconate. N-.. Gluconic acid, sodium giuconate,

potassium gluconate, ammonium giuconate.

In Table XII to XXI, inclusive, the concentrations in the left-handcolumns are weight of solids calculated as sodium -gluconate per totalweight of the solution. The symbols have the following meanings:

L= Clear liquid solution LLLLLLLLL LLLLLLLLL LLLLLLLLL LLLLLLLLLLLLLLLLLL LLLLLLLLL LLLLLLLLL Observations after indicated days at 57 C.

TABLE XVI.COMPOSITION E Concentration, percent:

LLLLTSSSS LLLLLSSSS LLLLLSSSS LLLLLSSSS LLLLLSSSS LLLLLCSSS Observationsafter indicated days LLLLLLLLL t d E m m L o m w T wa am mew mwl i TCSConcentration, percent:

LLLLLLLLL LLLLLLLLL LLLLLLLLL LLLLLLLLL LLLLLLLLL LLLLLLLLL LLLLLLLLL qn n n 5 0 5 0 5 0 5 66677889 Observations after indicated days at 5-7 C.

TABLE XVII.-COMPOSITION F Concentration, percent:

LLLLLLLsL Observations after indicated days at 57 C.

TABLE XIIL-COMPOSITION B Concentration, percent:

LLLLLLLLL LLLLLLLLL LLLLLLLLL LLLLLLLLL C LLLLLLLLL LLLLLLLLLM LLLLLLLLLLLLLLLLSL LLLLLTTLL LLLLLLLLF LLLLLLLLLW LLLLLLLL W LLLLLLLLLM TAB LEXXIV-COMPOSITION M TABLE XVIIL-COMPOSITION G Observations afterindicated days at Observations after indicated days 40 Concentration,perccnt z Concentration, percent:

LLLLLCTTT LLLLLLLLL LLLLLLLLL LLLLLLLLL LLLLLLLLL LLLLLL LLLSSSS LLLCSSSLLLTSSS LLLLSSS LLLLSSS LLLLSSS LLLLLLL TABLE XIX.COMPOSITION H 1 Asaverage sodium gluconate plus ammonium gluconate equivalent.

TABLE XXV.-COMPOSITION N Observations after indicated days at fi7 C.

15 Observations after indicated days at Concentration, percent:

LLLLLLLLL LLLLLLLLL LLLLLLLLL LLLLLLLLL 5 LLLLLLLLL Concentration,percent z LLLLLLLLL LLLLLLLLL LLLLLLLLL LLLLLLLLL LLLLLLLLL o LLLLLLLLLAs average sodium gluconate plus potassium gluconate plus am- TABLEXX.COMPOSITION I monium gluconate equivalent Observations afterindicated days at EXAMPLE 8 (a) Filtrate from a fermentation carried outand processed according to Examples 4 and 5 and containingConcentration, percent:

LLLLL 30 36.3% gluconic acid and 60.4% sodium gluconate equivalent(gluconic acidzsodium gluconate ratio=1.81) was passed through a 5.5 cm.diameter glass column containing 1 liter of Dowex 50-X10 resin in thehydrogen form until the efliuent tested positive for sodium. At thispoint, 2500 ml. of filtrate solution had been fed to the columnLLLLLSTTT LLLLLLLLL LLLLLLLLL LLLLLLLLL LLLLLLLLL LLLL (followed by awater wash), and it was found that the TABLE XXL-COMPOSITION .T gluconicacid content in the total efiluent had increased from 906 grams to 1284grams, of which 996 grams of dium-free in the first efllu- -freeefliuent solution was very light colored and contained the gluconic acidin essentially 100% purity.

Observations after indicated days at 57 C.

gluconic acid were recovered so ent out. This recovered sodiumConcentration, percent:

(b) To show the advantage of using low sodium content gluconicacid-gluconate mixtures for the recovery of gluconic acid, the resinpurification was repeated using a mixture containing a higherconcentration of sodium. In this example, filtrate from a fermentationcarried out and processed according to Examples 4 and 5 and containingLLLLLLLLL LLLLLLLLL LLLLLLLLL LLLLLLLLL LLLLLLLLL LLLLLLLLL 26.1%gluconic acid and 58.0% sodium gluconate equivalent (gluconicacidzsodiurn gluconate ratio 0.90) was passed through a 5.5 cm. diameterglass column containing 1 liter of Dowex 50-X-1O resin in the hydrogenform until the effiuent tested positive for sodium. At this point,

Concentration, percent 1850 ml. of filtrate solution had been fed to thecolumn (followed by a water wash), and it was found that the gluconicacid content in the total efiluent had increased from 483 grams to 889grams, of which 660 grams of gluconic acid were recovered sodium-free inthe first eflluent cut. This recovered sodium-free efiiuent solution wasvery light colored and contained the gluconic acid in essentially 100%purity.

LLLLLLLLL LLLLLLLLL LLLLLLLLL LLLLLLLLL LLLLLLLLL As potassium gluconateequivalent.

EXAMPLE 9 Clear filtrate from a fermentation car essed according toExamples 4 and TABLE XXIIL-COMPOSITION L ried out and proc- 8 andcontaining gluconlc acid tested positive for sulfate, phosphate,

Observations after indicated days at 57 C.

30 oxalate, etc. It was slurried with calcium carbonate (0.7%) anddiatomaceous earth (1% filtrate contained 42.2% for sulfate, phosphate,oxalate, etc.

and filtered. The gluconic acid and tested negative EXAMPLE 10 Thesequestering power of the sodium gluconate dry product and gluconicacid-sodium gluconate liquid prodnot toward calcium ions was measured bythe Zussman LLLLLLLLL LLLLLLLLL LLLLLLLLL LLLLLLLLL LLLLLLLLL 1 Asammonium gluconate equivalent.

15 method (Soap Sanit. Chemicals, 24, 57 1948)) as described anddiscussed by Mehltretter et al. (Ind. Eng. Chem, 45, 2. 782 (1953)). Inaddition, the sequestering power of these compositions toward iron wasmeasured by the method described and discussed by Mehltretter.

(a) A sodium gluconate dry product produced according to Examples 4 and6 and having a purity of 96 .6% sequestered 16.1 grams of calcium and319 grams of iron per 100 grams of sodium gluconate following theprocedures described above.

(b) A gluconic acid-sodium gluconate liquid product containing 62.2% byweight sodium gluconate equivalent (gluconic acidzsodium gluconateratio-=0.6 7) produced according to Example 4 sequestered 16 grams ofcalcium and 319 grams of iron per 100 grams of sodium gluconateequivalent following the procedures described above.

EXAMPLE 11 The gluconic acid-sodium gluconate liquid product producedaccording to Example 4 and containing 60.7% by weight sodium gluconateequivalent (gluconic acidsodium gluconate ratio=0.84) was used in acommercial caustic bottle washing operation. The water source for thesoaker compartments had a hardness (as CaCO of approximately 350 p.p.m.,and a 3-5% caustic solution was used for the bottle washing operation.The gluconic acid-sodium gluconate liquid product was added to thecaustic solution at the rate of 8 pounds (4.85 pounds of sodiumgluconate equivalent) per 100 pounds of caustic solids. Excellent bottlewashing results were obtained with efficient removal of the aluminumlabels and the bottles after treatment showed no haze or rust spots.

EXAMPLE 12 Sodium gluconate dry product (0.5 pound, 96% pure), producedaccording to Examples 4 and 6, and caustic soda (1.5 pounds) were addedto 1 gallon water and the mixture heated to boiling. A rusted iron panelstrip (2" x 3%") cut from a rusted carbon steel sheet was half immersedin the boiling solution. After minutes, the iron strip was removed,rinsed with water and the treated half of the iron strip compared to theuntreated portion. The procedure was repeated using pieces cut fromdifferent portions of the rusted iron sheet. Excellent rust removal wasobtained.

From the foregoing description and examples it will be seen that theinvention provides a very versatile and flexible process for makingaldonic acid and aldonates, more particularly gluconic acid andgluconates and mixtures thereof, from aldoses such as glucose. Otheraldonic acids and aldonates are arabonic acid, mannonic acid, gulonicacid, galactonic acid and talonic acid and their salts.

The invention does not reside in the particular microorganism used inthe fermentation. The organism should be one capable of producing analdose oxidase, e.g., glucose oxidase, under agitated submerged aerobicconditions. Fungal organisms are preferred, especially Aspergillusniger. Other examples are Penicillium leuteum' and acid producingbacteria such as those of the genus Acetobacter. Nor does the inventionreside in the particular conditions except that conditions which promotecell growth of the organisms should be used including aeration with airand/or oxygen under submerged conditions, at atmospheric orsuperatmospheric pressures, in the presence of nitrogen and nutrientminerals such as magnesium, potassium and phosphate, and temperaturesusually within the range of 25 C. to 40 C. As previously indicated,however, pH of the medium is an important factor in the early stages offermentation and a pH of 5 to 7 is desirable in this stage. The timeperiod used is normally sufficient to convert nearly all of the aldoseto the aldonic acid or aldonate. A preferred temperature is about 34 CLThe isolated crude or purified aldose oxidahe enzyme system, oralternatively the microbial cells containing the aldose oxidase enzymesystem, can also be used to produce the aldonic acid and aldonates asherein described.

The preferred products consist of mixtures in which the acid: saltweight ratio is within the range of 0.2:1 4:1. In the case of gluconicacid-sodium gluconate mixtures, the preferred products are aqueoussolutions having a dissolved solids content in excess of 50% but usuallynot more than by weight, with a gluconic acid: sodium glutonate weightratio of 0.5,1 to 3:1. Mixtures of gluconic acid and other gluconateswould have similar ratios.

Control of pH during fermentation is effected by adding an alkali orbase, e.g., sodium hydroxide, potassium hy' droxide, or ammonia,continuously or intermittently, preferably as concentrated solutions inwater. The amount of alkali or base added preferably is sufficient toconvert at least one-third of the weight of aldonic acid formed to analdonate. Thereafter, the process can be operated with or without pHcontrol, depending upon the ratio of aldonic acid to aldonate desired inthe final product.

The microorganism utilizes small amounts of the aldose for cell growthand the formation of the aldose oxidase. After cell growth has beenestablished the additional aldose can be added as a solid or indissolved state continuously or intermittently. The addition of aldosein solid form is preferred because it does not substantially alter thevolume of the medium.

It will be seen that the process affords a number of variations. Thus,glucose and sodium hydroxide can be added to the fermentation medium inproportions such that the final product is essentially a solution ofsodium gluconate which is filtered to remove mycelium and other solids.The filtrate containing the sodium gluconate can be used as such or thesodium gluconate can be recovered therefrom. Alternatively, thefermentation medium can also be treated with small amounts of a calciumor barium salt which will form water insoluble phosphates, oxalates andsulfates, and thereafter filtered to remove the phosphates, oxalates andsulfates in the filter cake. The resultant filtrate contains the sodiumgluconate Without sulfate, oxalate and phosphate impurities.

When the process is carried out to produce a mixture of aldonic acid andaldonate, the mixture can be sold and used as such, or it can beneutralized to form an aldonate solution, which can be used as such orevaporated to produce a solid salt of 96-98% purity. The aldonate canalso be crystallized from solution to produce a product of 100% purity.

The mixtures of aldonic acid and aldonate in aqueous solution normallyhave a pH less than 5. Thus, the gluconic acid-gluconate=mixture canhave a weight ratio of gluconic acid to gluconate of 0.06:1 to 44:1. Ata ratio of 44:1 the pH is approximately 1.7.

Another modification is to produce a mixture of aldonate and aldonicacid in the manner previously described, e.g., a 60% solution (60 gramsper 100 cc. of solution) of gluconic acid and sodium gluconate and passsaid solution through a cation resin in hydrogen form (e.g., Dowex 50,Nalcite HCR, or other sulfonated styrene-divinylbenzene ion exchangeresin in the hydrogen form) thereby removing the sodium ions andconverting the sodium gluconate to gluconic acid which can be used assuch or converted to glucono-delta-lactone.

In another modification a mixture of aldonic acid and aldonate isprepared as previously described and is passed through a dialysismembrane or an electrodialysis cell where the acid, or part of it forexample, gluconic acid, is separated from the salt, for example, sodiumgluconate, thereby producing a gluconic acid solution and a sodiumgluconate solution.

The products in the form of aldonic acid-aldonate mixtures or as theacid or aldonate are useful in cleaning solutions, especially alkalinebottle washing solution, as chelating agents and for many otherpurposes.

The invention is hereby claimed as follows:

1. In the preparation of gluconic acid and gluconates by submergedaerobic fermentation of glucose in a fermentation medium containingmicroorganisms and nutrients capable of converting said glucose togluconic acid, the improvement which comprises adding a portion of saidglucose to said fermentation medium initially, fermenting at least apart of said portion at a pH of to 7, adding and fermenting additionalglucose in said medium and after the addition of said additional glucoseallowing the pH to drop below 5 due to the formation of gluconic acid.

2. In the preparation of gluconic acid and gluconates by submergedaerobic fermentation of glucose in a fermentation medium containingmicroorganisms and nutrients capable of converting said glucose togluconic acid, the improvement which comprises adding a portion of saidglucose to said fermentation medium initially, fermenting at least apart of said portion at a pH of 5 to 7, and adding and fermentingadditional such glucose in said medium, the total amount of g ucoseadded including the initial amount being in excess of 40% of thefermentation medium.

3. A process as claimed in claim 1 in which the pH of the fermentationmedium is initially controlled within a pH of 5 to 7 and after theaddition of said additional glucose the pH is allowed to drop below 5due to the formation of gluconic acid.

4. A process as claimed in claim 1 in which at least one neutralizingagent is added to the fermentation medium which forms a gluconate andalso forms water soluble sulfates, oxalates, and phosphates and when thefermentation is concluded a quantity of an alkaline earth metal compoundis added in sufiicient amount to form water insoluble sulfates, oxalatesand phosphates which are separated from said medium.

5. A process as claimed in claim 4 in which said alkaline earth metalcompound is calcium carbonate.

6. A process as claimed in claim 4 in which said alkaline earth metalcompound is calcium hydroxide.

7. In the preparation of gluconic acid and gluconates by submergedaerobic fermentation in a fermentation medium containing microorganismsand nutrients capable of converting glucose to gluconic acid, theimprovement which comprises adding glucose to the fermentation mediuminitially in an amount less than 40% of the medium, thereafterfermenting the medium while maintaining a pH of 5 to 7 at least in theearly stages of the fermentation, adding additional glucose to thefermentation medium until the total amount of glucose added includingthe initial amount is in excess of 40% of the fermentation medium, andfermenting the added glucose.

8. In the preparation of gluconic acid and gluconates by submergedaerobic fermentation in a fermentation medium containing microorganismsand nutrients capable of converting glucose to gluconic acid, theimprovement which comprises adding glucose to the fermentation mediuminitially in an amount less than 40% of the medium, thereafterfermenting the medium initially at a pH of 5 to 7, adding additionalglucose to the fermentation medium until the total amount of glucoseadded including the initial amount is between %to of the fermentationmedium, and fermenting the added glucose.

9. In the preparation of gluconic acid and gluconates by submergedaerobic fermentation in a fermentation medium containing microorganismsand nutrients capable of converting glucose to gluconic acid, theimprovement which comprises adding glucose to the fermentation mediuminitially in an amount less than 40% of the medium, thereafterfermenting the medium with the addition of an alkali suflicient tomaintain a pH of 5 to 7, adding additional glucose to the fermentationmedium until the total amount of glucose added including the initialamount is in excess of 40% of the fermentation medium, and fermentingthe added glucose.

10. A process as claimed in claim 9 in which said alkali comprisessodium hydroxide.

11. A process as claimed in claim 9 in which said alkali comprisespotassium hydroxide.

12. A process as claimed in claim 9 in which said alkali comprisesammonium hydroxide.

13. In the preparation of gluconic acid and gluconates, the improvementwhich comprises fermenting glucose in a fermentation medium containingmicroorganisms and nutrients capable of converting said glucose togluconic acid while maintaining a pH of 5 to 7 until said microorganismshave produced substantially optimum amounts of cell growth and glucoseoxidase, adding additional glucose to said medium and continuing thereaction while allowing the pH to drop below 5.

- References Cited UNITED STATES PATENTS 2,602,768 7/1952 Crocker et a1.36 2,651,592 9/1953 Baker 195--36 LIONEL M. SHAPIRO, Primary ExaminerU-.$- X-R. 19511 7 mg?" UNITED STATES PATENT OFFICE CERTIFICATE OFCORRECTION Patent No. 3,576,718 Dated Ap 7 97 Invenmfl Jaok Ziffer et a1It is certified that error appears in the above-identified patent andthat said Letters Patent are hereby corrected as shown below:

Column 1, line 4 "use" should read uses Column 2, line 26, "glusenic"should read gluoonic Column 9, Table 8, opposite "Final pH", "6.4 O 3.2"should Column 11, line +0, "Table" should read Tables Column 11, TableXIII, under "20", opposite "75", "T" should read L -a opposite "T"should read L opposi 5", "L" should read T Column 14, line +1, "out"should read cut lines 68 ar "The filtrate" should read The clearfiltrate Column 15, line 60, "leuteum" should read luteum Column 16,line 1, "oxidahe" should read oxidase line 6 and 7, "0.2:1 4:1" shouldread 0.2: l to l line 11 "0.5,1" should read 0.5:1

Signed and sealed this 214th day of August 1971.

(SEAL) Attest:

EDWARD M.FLETCHER,JR. WILLIAM E. SGHUYLER, JR. fttesting OfficerCommissioner of Patents

